Axonemal Doublet Microtubules Research Articles

Heart of the Beat (the Flagellar Beat, that Is)

Heart of the Beat (the Flagellar Beat, that Is)

Coordination of outer arm dynein activity along axonemal doublet microtubules

This study considers the mechanism by which ODA based sliding is produced and the relationship of that mechanism to the determination of beat frequency. Two models of activity have been examined: a stochastic model, where ODA activity is random and a metachronal model, where activity is sequentially triggered along a doublet. Inactivation of a few ODAs would have virtually no effect on stochastic activity, but would completely block metachronal activity. We (Seetharam and Satir [2005]: Cell Motil Cytoskeleton 60:96-103) previously demonstrated that ODAs produce high speed sliding of about 200 mum/s, followed by a pause. IDAs produce slow, 5 mum/s, continuous sliding. We have examined the effects of nM concentrations of vanadate on sliding, measuring velocity and extent of high speed sliding and pause distribution or sliding cessation. In 5 nM vanadate, where photocleavage experiments show about 16/270 ODAs per doublet are affected, no differences from control are seen, but at 10 and 25 nM vanadate, high speed velocity is greatly reduced and pause distribution changes. The results support a model, in which high speed sliding is produced by metachronal activity. Blockage of two or more heavy chains of one ODA or a small group of adjacent ODAs produces cessation of sliding, but cessation is only temporary, probably because IDA activity continues, allowing ODA activity re-initiation beyond the block. These conclusions are consistent with Sugino and Naitoh's [1982; Nature 295:609-611] proposal, whereby during each beat, every ODA along a doublet becomes activated in succession, with repetitive activation determining beat frequency.

Spermatotoxic effect of aflatoxin B1 in rat: extrusion of outer dense fibres and associated axonemal microtubule doublets of sperm flagellum

Male Wistar rats were treated with aflatoxin B1 (AFB1). Live as well as methanol-fixed cauda epididymal spermatozoa were stained with acridine orange (AO) and ethidium bromide (EB) and observed under a fluorescence microscope. Giemsa-stained smears were observed in a bright field microscope. Unstained smears were observed with phase contrast illumination. The axoneme of more than 10% of the spermatozoa of treated rats had the outer dense fibres (ODFs), in varying numbers, and the associated axonemal microtubule doublets of the flagellum extruded either at midpiece-principal piece junction or connecting piece. This could be perceived in all light microscopic preparations, but AO-EB staining offered an advantage of the assessment of the viability as well. TEM observation of sections of the testis and cauda epididymidis also revealed ODF extrusion, as seen in the transverse sections of sperm flagella missing one or more ODFs and the associated axonemal microtubule doublets. In a few such sections, the extruded elements were seen in the cytoplasm, outside the mitochondrial sheath or peripheral sheath. Marginal to severe mitochondrial pathologies were observed in the spermatozoa and elongated spermatids, suggesting a link between AFB1-induced sperm mitochondrial pathology and extrusion of ODFs. However, the possibility that AFB1 treatment would disrupt the cytoskeletal proteins of the flagellum, resulting in the extrusion of ODFs, cannot be excluded. This sperm abnormality is reported for the first time as produced by a dietary toxin. Dietary aflatoxins, therefore, could also be contributory factors for the deterioration of the reproductive health of men.

Kinetic model for dynein oscillatory activity

A kinetic model for dynein, a molecular motor, is considered. This model explains the oscillatory behaviour, observed by Chikako Shingyoji et al. [Ch. Shingyoji, H. Higuchi, M. Yoshimura, E. Katayama, T. Yanagida, Dynein arms are oscillatory force generators, Nature 393 (1998) 711–714.] and by Susumu Aoyama and Ritsu Kamiya [S. Aoyama, R. Kamiya, Cyclical interactions between two outer doublet microtubules in split flagellar axonemes, Biophys. J. 89 (2005) 3261–3268.] in surprisingly simple axonemal fragments. The model shows that sustained oscillations can be generated due to the obligate cooperative interaction of the two dynein heads in the axonemal fragments. No other feedback control interactions are involved in the model to explain oscillations, similar to those observed experimentally, for realistic dynein rate constants. The modified model shows how the ATP hydrolytic exhaustion influences the amplitude and frequency of dynein oscillatory activity.

219. Parkin co-regulated gene (Pacrg) is an axonemal protein involved in sperm tail and ependymal cell function and is a candidate primary ciliary dyskinesia gene

A leading cause of male infertility is genetic variation in genes required for sperm formation or function. Considerable evidence suggests PACRG is involved in spermiogeneis. The loss of Pacrg function causes infertility in mice (Lorenzetti et al. 2004) and we have shown an association between variability in the 5′ untranslated region of PACRG and human male infertility (Wilson et al. in preparation). Evidence from studies in C.reinhardtii and T.brucei indicate Pacrg is crucial for axonome formation and microtubule stability. To assess this possibility in mammals, we generated and characterised Pacrg knockout (Quaking viable, Qkv), wildtype and Pacrg transgenic mice (Qkv-Tg). Using confocal and immunoelectron microscopy we showed that Pacrg was localised to the axonemal microtubule doublets of sperm, tracheal and ependymal cilia. The absence of Pacrg was associated with compromised sperm flagella formation and MRI analyses revealed the occurrence of hydrocephalus. Specifically, Qkv mice showed an inward expansion of the lateral ventricles, resulting in a significant reduction in distance between ventricles (1.0 ± 0.6 mm, mean ± s.d., n = 5) and a ~250% increase in ventricle area (70 ± 13 arbitrary units, mean ± s.d., n = 5) compared with wildtype littermates (1.38 ± 0.09 mm; area 26 ± 12, n = 3). Transgenic expression of Pacrg was necessary and sufficient to correct the hydrocephalus (1.45 ± 0.05 mm; area 26 ± 9, n = 2) and infertility phenotypes (evidenced by daily sperm counts and litter sizes). In conclusion, we have shown Pacrg is a novel axoneme associated protein in a subset of motile cilia/flagella and loss of Pacrg function results in spermiogenic defects and hydrocephalus in mice. Further, we have shown that variations in the human PACRG promoter are a risk factor in human male infertility. Collectively these data suggest PACRG is a candidate gene in the human syndrome of primary ciliary dyskinesia. (1) Lorenzetti D, Bishop CE, Justice MJ. 2004. Deletion of the Parkin coregulated gene causes male sterility in the quaking (viable) mouse mutant. Proc Natl Acad Sci U S A 101(22):8402–8407

Molecular architecture of axonemal microtubule doublets revealed by cryo-electron tomography

The axoneme, which forms the core of eukaryotic flagella and cilia, is one of the largest macromolecular machines, with a structure that is largely conserved from protists to mammals. Microtubule doublets are structural components of axonemes that contain a number of proteins besides tubulin, and are usually found in arrays of nine doublets arranged around two singlet microtubules. Coordinated sliding of adjacent doublets, which involves a host of other proteins in the axoneme, produces periodic beating movements of the axoneme. We have obtained a three-dimensional density map of intact microtubule doublets using cryo-electron tomography and image averaging. Our map, with a resolution of about 3 nm, provides insights into locations of particular proteins within the doublets and the structural features of the doublets that define their mechanical properties. We identify likely candidates for several of these non-tubulin components of the doublets. This work offers insight on how tubulin protofilaments and accessory proteins attach together to form the doublets and provides a structural basis for understanding doublet function in axonemes.

Cyclical Interactions between Two Outer Doublet Microtubules in Split Flagellar Axonemes

The beating of cilia and flagella is based on the localized sliding between adjacent outer doublet microtubules; however, the mechanism that produces oscillatory bending is unclear. To elucidate this mechanism, we examined the behavior of frayed axonemes of Chlamydomonas by using high-speed video recording. A pair of doublet microtubules frequently displayed association and dissociation cycles in the presence of ATP. In many instances, the dissociation of two microtubules was not accompanied by noticeable bending, suggesting that the dynein-microtubule interaction is not necessarily regulated by the microtubule curvature. On rare occasions, association and dissociation occurred simultaneously in the same interacting pair, resulting in a tip-directed movement of a stretch of gap between the pair. Based on these observations, we propose a model for cyclical bend propagation in the axoneme.

Solution Structure of the Tctex1 Dimer Reveals a Mechanism for Dynein-Cargo Interactions

Tctex1 is a light chain found in both cytoplasmic and flagellar dyneins and is involved in many fundamental cellular activities, including rhodopsin transport within photoreceptors, and may function in the non-Mendelian transmission of t haplotypes in mice. Here, we present the NMR solution structure for the Tctex1 dimer from Chlamydomonas axonemal inner dynein arm I1. Structural comparisons reveal a strong similarity with the LC8 dynein light chain dimer, including formation of a strand-switched beta sheet interface. Analysis of the Tctex1 structure enables the dynein intermediate chain binding site to be identified and suggests a mechanism by which cargo proteins might be attached to this microtubule motor complex. Comparison with the alternate dynein light chain rp3 reveals how the specificity of dynein-cargo interactions mediated by these dynein components is achieved. In addition, this structure provides insight into the consequences of the mutations found in the t haplotype forms of this protein.

Rat Spag5 associates in somatic cells with endoplasmic reticulum and microtubules but in spermatozoa with outer dense fibers

The leucine zipper motif has been identified as an important and specific interaction motif used by various sperm tail proteins that localize to the outer dense fibers. We had found that rat Odf1, a major integral ODF protein, utilizes its leucine zipper to associate with Odf2, another major ODF protein, Spag4 which localizes to the interface between ODF and axonemal microtubule doublets, and Spag5. The rat Spag5 sequence indicated a close relationship with human Astrin, a microtubule-binding spindle protein suggesting that Spag5, like Spag4, may associate with the sperm tail axoneme. RT PCR assays indicated expression of Spag5 in various tissues and in somatic cells Spag5 localizes to endoplasmic reticulum and microtubules, as expected for an Astrin orthologue. MT binding was confirmed both in vivo and in in vitro MT-binding assays: somatic cells contain a 58 kDa MT-associated Spag5 protein. Western blotting assays of rat somatic cells and male germ cells at different stages of development using anti-Spag5 antibodies demonstrated that the protein expression pattern changes during spermatogenesis and that sperm tails contain a 58 kDa Spag5 protein. Use of affinity-purified anti-Spag5 antibodies in immuno electron microscopy shows that in rat elongated spermatids and epididymal sperm the Spag5 protein associates with ODF, but not with the axonemal MTs. This observation is in contrast to that for the other Odf1-binding, MT-binding protein Spag4, which is present between ODF and axoneme. Our data demonstrate that Spag5 has different localization in somatic versus male germ cells suggesting the possibility of different function.

Cloning of Chlamydomonas p60 katanin and localization to the site of outer doublet severing during deflagellation

Katanin, a heterodimeric microtubule-severing protein that localizes to sites of microtubule organization, can mediate in vitro the ATP-dependent disassembly of both taxol-stabilized microtubules and axonemal doublet microtubules. In the unicellular biflagellate alga Chlamydomonas, katanin has been implicated in deflagellation, a highly specific process that involves a Ca2+-signal transduction pathway starting at the plasma membrane and culminating in the severing of axonemal outer doublet microtubules and excision of both flagella from the cell body. Previously, we showed that the microtubule severing activity of deflagellation and katanin's 60 kD catalytic subunit (termed p60) purified with the flagellar basal body complex (FBBC). Additional evidence supporting the involvement of katanin in deflagellation came from the observation that an antibody against human p60 katanin significantly inhibited FBBC-associated microtubule-severing activity. Here we report the cloning of p60 katanin from Chlamydomonas reinhardtii. Immunogold electron microscopy places Chlamydomonas p60 at several locations within the basal body apparatus and associated structures. Importantly, we find a dense accumulation of colloidal gold labeling the distal end of the flagellar transition zone, the site of outer doublet severing during deflagellation. These results suggest that, in addition to a potential involvement in the deflagellation pathway, katanin-mediated microtubule-severing may be associated with multiple processes in Chlamydomonas. Cell Motil. Cytoskeleton 43:221–231, 1999. © 1999 Wiley-Liss, Inc.

Cloning of Chlamydomonas p60 katanin and localization to the site of outer doublet severing during deflagellation.

Katanin, a heterodimeric microtubule-severing protein that localizes to sites of microtubule organization, can mediate in vitro the ATP-dependent disassembly of both taxol-stabilized microtubules and axonemal doublet microtubules. In the unicellular biflagellate alga Chlamydomonas, katanin has been implicated in deflagellation, a highly specific process that involves a Ca(2+)-signal transduction pathway starting at the plasma membrane and culminating in the severing of axonemal outer doublet microtubules and excision of both flagella from the cell body. Previously, we showed that the microtubule severing activity of deflagellation and katanin's 60 kD catalytic subunit (termed p60) purified with the flagellar basal body complex (FBBC). Additional evidence supporting the involvement of katanin in deflagellation came from the observation that an antibody against human p60 katanin significantly inhibited FBBC-associated microtubule-severing activity. Here we report the cloning of p60 katanin from Chlamydomonas reinhardtii. Immunogold electron microscopy places Chlamydomonas p60 at several locations within the basal body apparatus and associated structures. Importantly, we find a dense accumulation of colloidal gold labeling the distal end of the flagellar transition zone, the site of outer doublet severing during deflagellation. These results suggest that, in addition to a potential involvement in the deflagellation pathway, katanin-mediated microtubule-severing may be associated with multiple processes in Chlamydomonas.

Rotation of the central pair microtubules in eukaryotic flagella.

SUMMARY It has been 20 years since the phenomenon of centralpair rotation in eukaryotic flagella was reported(Omoto, and Kung, 1979). At that time, a model wasproposed in which the central pair functions as adistributor to regulate dynein activity among the outerdoublet microtubules. The video evidence obtainedsince then and gathered together in this essay is con-sistent with this model. Geometric arguments indicatethat there must be circumferential and longitudinalregulation of shear forces to produce effective bendingmotion of an axoneme. Central pair microtubules areideally situated to perform this regulatory function.The regulation of outer doublet sliding by the centralpair, together with the rotation of the latter, where thisoccurs, may explain the wide diversity of two- andthree-dimensional flagellar and ciliary waveforms thatis found in organisms using the same basic 9 1 2structure. ACKNOWLEDGMENTS We thank Mike McLaughlin of Material Media Services, Washing-ton State University for converting 16-mm film to video format, andDenise A. Palmen (Technical Services, Washington State University)for editing the videos, assembling the QuickTime movies, and pro-ducing the jpeg images to use as figures. The research based onvideo images shown in this essay was first published by Kamiya(1982), Omoto and Witman (1981), Gibbons

Direct measurement of inter-doublet elasticity in flagellar axonemes.

The outer doublet microtubules in ciliary and flagellar axonemes are presumed to be connected with each other by elastic links called the inter-doublet links or the nexin links, but it is not known whether there actually are such elastic links. In this study, to detect the elasticity of the putative inter-doublet links, shear force was applied to Chlamydomonas axonemes with a fine glass needle and the longitudinal elasticity was determined from the deflection of the needle. Wild-type axonemes underwent a high-frequency, nanometer-scale vibration in the presence of ATP. When longitudinal shear force was applied, the average position of the needle tip attached to the axoneme moved linearly with the force applied, yielding an estimate of spring constant of 2.0 (S.D.: 0.8) pN/nm for 1 microm of axoneme. This value did not change in the presence of vanadate, i.e., when dynein does not form strong cross bridges. In contrast, it was at least five times larger when ATP was absent, i.e., when dynein forms strong cross bridges. The measured elasticity did not significantly differ in various mutant axonemes lacking the central-pair microtubules, a subset of inner-arm dynein, outer-arm dynein, or the radial spokes, although it was somewhat smaller in the latter two mutants. It was also observed that the shear displacement in an axoneme in the presence of ATP often took place in a stepwise manner. This suggests that the inter-doublet links can reversibly detach from and reattach to the outer doublets in a cooperative manner. This study thus provides the first direct measure of the elasticity of inter-doublet links and also demonstrates its dynamic nature.

Unidirectional movement of fluorescent microtubules on rows of dynein arms of disintegrated axonemes.

Tetramethylrhodamine-labelled microtubules were observed to move on rows of dynein arms of sea urchin sperm axonemes exposed by elastase-induced sliding disintegration. The microtubules moved towards the flagellar tip at a velocity of 3.1+/-2.1 microm second-1 (mean +/- s.d., n=53) in the presence of 0.1 mM ATP at 22 degrees C, but none moved towards the sperm head. We also examined the polarity of microtubule binding to axonemal doublet microtubules in the absence of ATP by using microtubules brightly labelled at their minus-ends. In 140 of 210 microtubules studied, they bound to axonemal microtubules with a parallel polarity. These results suggest that tightly packed dynein arms on the outer doublet microtubules of sperm axoneme preferentially bind microtubules to themselves with the same polarity as that of the axoneme and that they generate a force to move only these microtubules in the direction away from the sperm head.

Leishmania major: In vitro ultrastructural study of the paraxial rod of promastigotes

The flagellum of Leishmania major promastigotes has an intraflagellar structure known as the paraxial rod (PAR) which extends from a point halfway in the flagellar pocket to the tip of the flagellum, lying opposite the axonemal microtubule doublets 4–7. An expansion of the axonemal plasma membrane envelops the PAR and may provide desmosomal attachment at the orifice of the flagellar pocket. The complex organization of the 4–6 nm thick filaments in the PAR was studied by us in cross, oblique, longitudinal and tangential sections by electron microscope. These filaments are disposed in two parallel lamellae, one alongside the axoneme ( ca. 45 nm thick), and the other alongside the plasma membrane ( ca. 65 nm thick), with an interlamellar gap of about 22–28 nm. In each lamella, 8–12 parallel series of longitudinal filaments at ca. 30 nm intervals interdigitate with coplanar parallel series of oblique filaments at ca. 25 nm intervals and inclined to the long axis of the flagellum at ca. 48°, and ca. 55°, in the inner (paraxonemal) and outer lamella, respectively. The parallel filaments in each of the longitudinal and oblique series are spaced at ca. 8 nm intervals. They are cross-striated at ca. 30 nm intervals by transverse filaments which terminate occasionally on adjacent axonemal microtubules 5 and 6 in the inner lamella, and the plasma membrane in the outer lamella. Extending across the interlamellar gap is a set of parallel rows of 7–12 nearly parallel filaments at ca. 20 nm intervals. The part of the flagellar plasma membrane enclosing the PAR has a subplasmalemmal cytoskeleton consisting of a layer of longitudinal 2 nm filaments at 8 nm intervals, obliquely striated by parallel 2 nm filament doubles at ca. (−65)° with the long axis of the flagellum and ca. 20 nm periodicity. Each filament doublet stria apparently gives origin to collinear short filament doublet extensions that curve into juxtaposed meshes of the outer lamella. Microtubules of the axonemal doublets 5 and 6 are connected to electron-dense ( ca. 12 nm thick) strips of the inner lamella of the PAR by longitudinal series of ca. 4 nm cross-links across a ca. 12 nm cleft.

Structural and functional reconstitution of inner dynein arms in Chlamydomonas flagellar axonemes.

The inner row of dynein arms contains three dynein subforms. Each is distinct in composition and location in flagellar axonemes. To begin investigating the specificity of inner dynein arm assembly, we assessed the capability of isolated inner arm dynein subforms to rebind to their appropriate positions on axonemal doublet microtubules by recombining them with either mutant or extracted axonemes missing some or all dyneins. Densitometry of Coomassie blue-stained polyacrylamide gels revealed that for each inner dynein arm subform, binding to axonemes was saturable and stoichiometric. Using structural markers of position and polarity, electron microscopy confirmed that subforms bound to the correct inner arm position. Inner arms did not bind to outer arm or inappropriate inner arm positions despite the availability of sites. These and previous observations implicate specialized tubulin isoforms or nontubulin proteins in designation of specific inner dynein arm binding sites. Further, microtubule sliding velocities were restored to dynein-depleted axonemes upon rebinding of the missing inner arm subtypes as evaluated by an ATP-induced microtubule sliding disintegration assay. Therefore, not only were the inner arm dynein subforms able to identify and bind to the correct location on doublet microtubules but they bound in a functionally active conformation.

Effects of acylpeptide K-26 on the motility of sea urchin sperm model. I. Inhibition of the motility of both live and triton X-100-extracted sperm.

An acylpeptide called K-26, which was isolated from a culture filtrate of Bacillus sp. 503, inhibited motility of both live sea urchin sperm as well as Triton X-100-extracted sperm model. In each case, complete inhibition was observed at concentrations of more than 1 μM or 6 μM, respectively. This inhibitory effect of K-26 on the Triton-extracted sperm could not be reversed by reducing the concentration of K-26 in the reactivation medium containing 1 mM ATP. However, it was reversed when the ATP concentration in the reac-tivation medium was simultaneously reduced.At concentrations below 4 μM, K-26 did not completely suppress the flagellar movement of the Triton-extracted sperm, but reduced the beat frequency in a competitive manner; the maximal beat frequency (Fmax) was 31.2 Hz whether in the absence or in the presence of 4 μM K-26, while Km for ATP was 0.19 mM or 0.28 mM, respectively. ATPase activity of isolated axonemes was increased severalfold in the presence of K-26. The sliding movement between the outer doublet microtubules in trypsin-treated axonemes was normal at K-26 concen-trations as high as 20 μM.From these results, it was suggested that K-26 affects the mechanism that con-verts the sliding movement between the outer doublet microtubules into a ben-ding motion, and thereby inhibits the motility of the Triton-extracted sperm model.

Evidence for tektins in centrioles and axonemal microtubules.

Affinity purified, polyclonal antibodies were prepared against three tektins (tektins A, B, and C) isolated from sea urchin sperm axonemal microtubules. These antibodies (anti-tektins) were used to localize tektins in axonemes, basal bodies, and centrioles. By immunofluorescence microscopy it could be demonstrated that in sperm tails from Lytechinus pictus all three anti-tektins stain all nine axonemal doublet microtubules and A-tubule extensions along their entire length. In addition to staining doublet microtubules, anti-tektin C weakly labeled the central-pair microtubules in sperm tails from Patiria miniata. The anti-tektin staining revealed also a clear cross-reaction with basal bodies of sea urchin sperm and with centrioles of cells from hamsters, humans, and pigs. These data provide evidence of tektin or tektin-like proteins in basal bodies and centrioles and suggest that centriole microtubules are constructed according to the same principles as flagellar microtubules.

Activation of sea urchin sperm flagellar dynein ATPase activity by salt-extracted axonemes.

When 21S dynein ATPase [EC 3.6.1.3] from sea urchin sperm flagellar axonemes was mixed with the salt-extracted axonemes, the ATPase activity was much higher than the sum of ATPase activities in the two fractions, as reported previously (Gibbons, I.R. & Fronk, E. (1979) J. Biol. Chem. 254, 187-196). This high ATPase level was for the first time demonstrated to be due to the activation of the 21S dynein ATPase activity by the axonemes. The mode of the activation was studied to get an insight into the mechanism of dynein-microtubule interaction. The salt-extracted axonemes caused a 7- to 8-fold activation of the 21S dynein ATPase activity at an axoneme : dynein weight ratio of about 14 : 1. The activation was maximal at a low ionic strength (no KCl) at pH 7.9-8.3. Under these conditions, 21S dynein rebound to the salt-extracted axonemes. The maximal binding ratio of 21S dynein to the axonemes was the same as that observed in the maximal activation of 21S dynein ATPase. The sliding between the outer doublet microtubules in the trypsin-treated 21S dynein-rebound axonemes took place upon the addition of 0.05-0.1 mM ATP in the absence of KCl. During the sliding, the rate of ATP hydrolysis was at the same level as that of the 21S dynein activated by the salt-extracted axonemes. However, it decreased to the level of 21S dynein alone after the sliding. These results suggested that an interaction of the axoneme-rebound 21S dynein with B-subfibers of the adjacent outer doublet microtubules in the axoneme causes the activation of the ATPase activity.

Helical conformation of dense fibres from mammalian spermatozoa

When dense (accessory) fibres are released from rat, hamster and rabbit spermatozoa by ultrasonic treatment, their fragments spontaneously adopt helical forms. The pitch of the helices is positively correlated with fibre thickness. The chirality is always sinistral. In becoming helical the fibres must undergo torsion, since the abaxial surface of the fibre always lies on the interior (concave) edge of the helix. A collapse or shortening of the cortical structure of the fibre would have this result. The helical shape cannot be explained by the bending behaviour of attached microtubular doublets, nor can it be merely a response peculiar to sonication. Sperm dense fibres develop as appendages to axonemal doublet microtubules and it is of interest that such doublets, when isolated from sea urchin spermatozoa, also form left-handed helices [23]. Either the axonemal complex is maintained under tension in vivo or the tendency to coiling is suppressed by readily dissociated components of the dense fibres.